Dysregulation of signal transduction pathways that control cell growth, differentiation, apoptosis, and motility is associated with many human pathologies. One of the key kinases involved in regulation of these pathways is the Protein Kinase C (PKC) family of isoenzymes. Because of the central role of these enzymes in signal transduction and human disease, the need for isoform-specific modulators of PKC activity - both for therapeutic and research purposes - is widely recognized as one of the major challenges in the field. The progress in this area has been significantly impeded by poor understanding of the molecular basis of PKC activation and regulation. Indeed, PKC presents significant challenges for conventional structural biology approaches due to its multi-modular structure, the associated inter-domain flexibility, and the amphiphilic nature of the N-terminal regulatory domain that undergoes membrane insertion upon enzyme activation. The long-term objective of my laboratory is to understand the molecular basis of activation of PKC isoforms through biophysical and biochemical studies of their most variable domains. The specific objective of this proposal is to characterize the key inter-domain and domain-cofactor interactions and test several novel hypotheses about their role in the activation process of the ? isoform of PKC. Our experimental approach makes an extensive use of the structural and functional autonomy of the PKC? domains and integrates advanced solution NMR techniques, fluorescence spectroscopy, X-ray crystallography, mutagenesis, and in-vitro membrane binding assays. The Specific Aims of this proposal are directed at (1) identifying the structural and functional interplay of lipid-binding domains essential for the PKC? membrane-insertion step and tumor-promoting response, and (2) testing the hypotheses that the C-terminal domain of PKC? serves as a membrane anchor and an intra-molecular protein interaction module. We anticipate that our findings will: generate insight into isoform-specific regulation of PKC? by identifying key residues involved in the interactions with membranes/membrane-embedded ligands and inter-domain interactions; provide a molecular platform for the design of isoform-specific agents that modulate the activity of PKC? through interference with its membrane binding and/or inter-domain interactions; and establish a structural framework for interpretation of in vitro and in viv functional data on conventional PKC isoforms.